Animal models of hemostasis are often extrapolated to humans; however, only a few studies have compared coagulation and fibrinolysis across species. Simultaneous thrombin (TG) and plasmin (PG) generation is useful to assessing coagulation and fibrinolysis within the same sample. In this study, we performed simultaneous TG and PG analysis in blood plasma samples from humans and 6 species commonly evaluated in pre-clinical research. TG and PG were investigated in male and female donor platelet-poor plasmas (PPP) obtained from 28 healthy humans, 10 baboons, 12 rhesus monkeys, 20 Yorkshire pigs, 20 Sprague-Dawley rats, 10 New Zealand White rabbits and 14 Hartley guinea pigs. The continuous generation of the 7-amino-4-methylcoumarin (AMC) from substrates specific to thrombin or plasmin was monitored. The thrombin and plasmin concentration peak heights (PH) and production rates (PR) were calculated. TG and PG parameters from baboon and rhesus macaque plasma approximated that of humans. The other species differed significantly from both human and non-human primates. For example, swine and rat plasmas demonstrated similar TG, but swine plasmas did not generate plasmin. TG and PG parameters from Guinea pig samples were extremely low, while rabbit plasmas showed variable PG curves demonstrating one or two peaks with low and high PR values, respectively. Correlations between PH and PR values were significant with the exceptions of human PG, baboon TG, rat TG and Guinea pig PG. These findings are informative to pre-clinical animal species selection and optimization of coagulation and fibrinolysis translational research.